Cell surface glycoprotein plays a role in the interactions that regulate many important biological processes, from cell-cell adhesion to signal transduction. Glycosylation is very common and important post-translational modification of proteins and has a critical role in cell-cell communication and recognition. The majority of proteins are either O-glycosylated (at serine or threonine residues) or N-glycosylated (at asparagine residues). Several reports have indicated that glycosylation can be altered in cancerous cells (Dwek et al., Proteomics 1:756-762, 2001). Protein glycosylation is abundant in extracelllular environments. These proteins include proteins on the extracellular side of the plasma membrane, secreted proteins and proteins contained in body fluids such as blood serum, cerebrospinal fluid, urine, breast milk, saliva, lung lavage fluid or pancreatic juice.
Typically, cell surface proteins are transmembrane proteins with the carbohydrate moieties on the outside of the cells. The capture of cell membrane proteins using different chemistries (i.e. reaction with free sulfhydryl groups, lysine groups) has been described (Hoffman, Clinical Chemistry 46: 1478-1486, 2000).
Periodate oxidation was first disclosed by Spiro (Spiro and Bhoyroo, J. Biol. Chem. 249: 5704, 1974). The method is to oxidize the carbohydrate moieties on a protein such as a glycoprotein. Sialic acids are the most abundant terminal components of oligosaccharides on mammalian cell-surface glycoproteins and are synthesized from the six-carbon precursor N-acetylmannosamine.
It is also well known in the art that the most common method for introducing aldehydes and ketones into polysaccharides and glycoproteins is by periodate-mediated oxidation of vicinal diols. Periodate oxidation has been used to develop tagged specific cell populations (Molecular Probe, OR, Product list 3.2. Hydrazines and Aromatic Amines for Modifying Aldehydes and Ketones). The cell surface sialic acid-rich glycoprotein has been described to be labeled by periodate/NaB3 H4 cell-surface labeling techniques (Spring et al, Biochem. J. 213:661-670, 1983). Moreover, periodate oxidation has been used for the oxidation of aldehyde groups on cell surface sialic acid at room temperature. Then these groups are used to ligate moieties onto the cell surface using hydrazides such as fluorescent hydrazide dye (Wood et al, European Cells and Mterials, 4, Suppl. 2:60-61, 2002.)
Cell surface proteins participate in sensing external signals and responding to environmental cues. Changes in the abundance of cell surface proteins can reflect a specific cellular state or the ability of a cell to respond to its changing environment. Therefore, the comprehensive, quantitative characterization of the protein components of the cell surface can identify marker proteins or a cluster of marker protein characteristics for a particular cellular state or explain the molecular basis for cellular responses to external stimuli. In many cases, changes in expression of a number of cell surface proteins such as Her2/Neu/erB, IGFI receptor and EFF receptor have been implicated in carcinogenesis and a current antibody therapy is based on targeting the cell surface protein Her2/neu receptor (Herceptin).
Isolated cell surface proteins depending upon enrichment of membranous subcellular fractions may result in substantial contamination with proteins that are localized to intracellular membranes (estimated to be around 15 to 30-fold more abundant than cell surface proteins). Other capture-based approaches rely upon the capture of cell surface proteins using non-covalent interactions. As this does not permit the stringent washing of immobilized proteins, a high degree of contamination occurs, which eliminates the empirical and broad identification and relative quantification of cell surface proteins. Therefore, it is important to obtain an enriched amount of pure cell surface protein, and to minimize “leaking” of cell content because numerous expressed proteins (present in the cytoplasm) are also glycosylated.